Modified gapmer oligomers and methods of use thereof

Abstract

The disclosure includes antisense oligonucleotides (ASOs), including gapmer ASOs, and methods of making and using the same.

1 . An antisense oligonucleotide (ASO), comprising 14-22 nucleotide units and:
 (a) a central region (B′) comprising 6 or more contiguous DNA nucleotides;   (b) a 5′-wing region (A′) comprising 2 to 6 locked nucleotides or 2′ substituted nucleosides; and   (c) a 3′-wing region (C′) comprising 2 to 6 locked nucleotides or 2′ substituted nucleosides;   wherein the central region of the ASO is at least 80% complementary or hybridizes to a target RNA sequence; and wherein the ASO comprises at least one modified nucleotide selected from:   
       
         
           
           
               
               
           
         
       
     
     
         2 . The ASO of  claim 1 , wherein (i) the central region (B′) comprises a modified nucleotide selected from G-clamp and 5prnl, (ii) the 5′-wing region (A′) comprises a modified nucleotide selected from Gutb and Nmln, (iii) the 3′-wing region (C′) comprises a modified nucleotide selected from Gutb and Nmln, or (iv) any combination thereof. 
     
     
         3 . The ASO of  claim 1 , wherein the central region (B′) comprises 2, 3, 4, 5, or 6 or more modified nucleotides. 
     
     
         4 . The ASO of  claim 1 , wherein the 5′-wing region (A′), the 3′-wing region (C′), or both comprise a modified nucleotide selected from Gutb, Nmln, G-clamp, and 5prnl. 
     
     
         5 . The ASO of  claim 1 , wherein the ASO molecule further comprises 1 or more phosphorothioate (ps) internucleoside linkages, mesyl phosphoroamidate (yp) internucleoside linkages, or a combination thereof. 
     
     
         6 . (canceled) 
     
     
         7 . An antisense oligonucleotide (ASO) comprising 14-22 nucleotide units, wherein the ASO comprises:
 (a) a central region (B′) comprising 6 or more contiguous DNA nucleotides, wherein at least one of the contiguous DNA nucleotides is a modified nucleotide,   (b) a 5′-wing region (A′) comprising 2 to 6 locked nucleotides or 2′ substituted nucleosides, and   (c) a 3′-wing region (C′) comprising 2 to 6 locked nucleotides or 2′ substituted nucleosides,   
       wherein the central region of the ASO is at least 80% complementary or hybridizes to a target RNA sequence, and wherein the ASO comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mesyl phosphoroamidate (yp) internucleoside linkages. 
     
     
         8 . The ASO of  claim 7 , wherein the ASO comprises at least 1, at least 2, at least 3, at least 4, or at least 5 or more nucleotide(s) selected from: 
       
         
           
           
               
               
           
         
       
     
     
         9 . The ASO of  claim 7 , wherein the ASO molecule further comprises 1 or more phosphorothioate (ps) internucleoside linkages 
     
     
         10 . The ASO of  claim 1 , wherein:
 (i) at least one mesyl phosphoroamidate (yp) internucleotide linkage is between nucleoside positions 3 and 4 from the 5′ end of the ASO molecule;   (ii) at least one mesyl phosphoroamidate (yp) internucleotide linkage is between nucleoside positions 5 and 6 from the 5′ end of the ASO molecule;   (iii) at least one mesyl phosphoroamidate (yp) internucleotide linkage is between nucleoside positions 6 and 7 from the 5′ end of the ASO molecule;   (iv) at least one mesyl phosphoroamidate (yp) internucleotide linkage is between nucleoside positions 7 and 8 from the 5′ end of the ASO molecule;   (v) at least one mesyl phosphoroamidate (yp) internucleotide linkage is between nucleoside positions 8 and 9 from the 5′ end of the ASO molecule;   (vi) at least one mesyl phosphoroamidate (yp) internucleotide linkage is between nucleoside positions 9 and 10 from the 5′ end of the ASO molecule; or   (vii) a combination thereof.   
     
     
         11 . The ASO of  claim 1 , wherein the 5′-wing region (A′), the 3′-wing region (C′), or both comprise at least one mesyl phosphoroamidate (yp) internucleotide linkage. 
     
     
         12 . The ASO of  claim 1 , wherein the ASO molecule further comprises a galactosamine. 
     
     
         13 . The ASO of  claim 12 , wherein the galactosamine is N-acetylgalactosamine (GalNAc) of Formula (VI): 
       
         
           
           
               
               
           
         
       
       wherein
 m is 1, 2, 3, 4, or 5; 
 each n is independently 1 or 2; 
 p is 0 or 1; 
 each R is independently H; 
 each Y is independently selected from —O—P(═O)(SH)—, —O—P(═O)(O)—, —O—P(═O)(OH)—, and —O—P(S)S—; 
 Z is H or a second protecting group; 
 either L is a linker or L and Y in combination are a linker; and 
 A is H, OH, a third protecting group, an activated group, or an oligonucleotide; or wherein the galactosamine is N-acetylgalactosamine (GalNAc) of Formula VII: 
 
       
         
           
           
               
               
           
         
       
       wherein R z  is OH or SH; and each n is independently 1 or 2. 
     
     
         14 . (canceled) 
     
     
         15 . The ASO of  claim 1 , wherein;
 (i) the target RNA sequence is a viral gene;   (ii) the target RNA sequence is a gene is from a DNA virus;   (iii) the target RNA sequence is a gene from a double-stranded DNA (dsDNA) virus;   (iv) the target RNA sequence is a gene from a hepadnavirus;   (v) the target RNA sequence is a gene from a hepatitis B virus (HBV);   (vi) the target RNA sequence is a gene from a HBV of any one of genotypes A-J; or   (vii) the target RNA sequence is selected from the S gene or X gene of a HBV.   
     
     
         16 . The ASO of  claim 1 , wherein the target RNA sequence is selected from a gene encoding a Methylation-Controlled J protein (MCJ protein), a gene encoding TAZ, a gene encoding angiopoietin like 3 (ANGPTL3), a gene encoding diacylglycerol acyltransferase 2 (DGAT2), and a gene encoding hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13). 
     
     
         17 . The ASO of  claim 1 , wherein (i) the 5′-wing region of the ASO comprises 2 to 6 phosphorothioate-linked locked nucleosides, (ii) the 3′-wing region of the ASO comprises 2 to 6 phosphorothioate-linked locked nucleosides, or (iii) a combination thereof, wherein the locked nucleosides are selected from LNA, ScpBNA, AmNA, AmNA (N-Me), GuNA, GuNA (N—R 11 ) where R 11  is selected from Me, Et, i-PR, t-Bu and combinations thereof. 
     
     
         18 . (canceled) 
     
     
         19 . The ASO of  claim 1 , wherein the central region of the ASO comprises at least 5 contiguous phosphorothioate-linked DNA nucleotides, at least 5 contiguous mesyl phosphoroamidate-linked DNA nucleotides, or at least 5 contiguous DNA nucleotides linked by one or more phosphorothioate internucleoside linkages and one or more mesyl phosphoroamidate internucleoside linkages. 
     
     
         20 . The ASO of  claim 7 , wherein the central region of the ASO comprises 8 to 10 contiguous phosphorothioate-linked DNA nucleotides, 8 to 10 contiguous mesyl phosphoroamidate-linked DNA nucleotides, or 8 to 10 DNA nucleotides linked by one or more phosphorothioate internucleoside linkages and one or more mesyl phosphoroamidate internucleoside linkages. 
     
     
         21 - 23 . (canceled) 
     
     
         24 . A pharmaceutical composition comprising the ASO of  claim 1 ; and a pharmaceutically acceptable excipient. 
     
     
         25 - 27 . (canceled) 
     
     
         28 . A method of treating a subject having a Hepatitis B virus (HBV) infection, comprising administering to the subject with HBV an ASO according to  claim 1 . 
     
     
         29 - 33 . (canceled) 
     
     
         34 . A method of decreasing expression of a target gene in a subject, comprising administering to the subject an ASO according to  claim 1 . 
     
     
         35 - 43 . (canceled)